Loss of functional enteric neurons results in clinical disorders such as achalasia, gastroparesis, neuropathic forms of pseudoobstruction, enteric neuronal dysplasia, colonic inertia and Hirschsprung's disease. Despite the lack of significant neurogenesis in vivo in these conditions, there is growing evidence to suggest the existence of multipotent neural precursor cells (NPCs) in abundant numbers in the gut. Several investigators including ourselves have shown that these cells, which have some phenotypical markers typical of glia, are capable of robust neurogenesis in vitro. We have now shown that enteric ganglia in longitudinal muscle myenteric plexus (LMMP) preparations display neurogenesis and this is even more pronounced in completely dissociated NPC preparations in culture. We have identified enteric neural precursors in vivo and in vitro and our preliminary studies implicate phosphatase and tensin homolog (PTEN) in these cells as the major brake on neurogenesis in vivo. Further collagen IV when added to LMMP preparations can have a profound inhibitory effect on neurogenesis. Thus it is logical to hypothesize that the discrepancy between the in vivo and ex vivo situation can be explained by factors in the microenvironment that regulate PTEN. In this proposal we therefore intend to examine the effects of collagen IV and downstream pathways including integrin, FAK and RhoA/ROCK which may drive PTEN activity, checking enteric neurogenesis in vivo, via the following specific aims: Specific Aim 1: To determine the interactions between extrinsic (collagen) and intrinsic (PTEN) brakes on neurogenesis in health and disease Specific Aim 2: To examine the effects of countering intrinsic and extrinsic brakes on enteric neurogenesis in vivo in health and disease Specific Aim 3: To examine the effects of manipulating selected key components of these pathways on survival and function of allogeneic enteric NSC (ENSC) transplants Our long-term objective is to develop neuronal transplantation as a treatment for human diseases of the enteric nervous system as well as stimulate neural regeneration in these disorders. By establishing a role for PTEN and related pathways the proposed studies will provide the critical groundwork for novel therapeutic interventions.